Frédéric Delbos

Multiple layers of B cell memory with different effector functions

Memory B cells are at the center of longstanding controversies regarding the presence of antigen for their survival and their re-engagement in germinal centers after secondary challenge. Using a new mouse model of memory B cell labeling dependent on the cytidine deaminase AID, we show that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M–positive (IgM+) and IgG1+ B cells in germinal center–like structures that persisted up to 8 months after immunization, as well as IgM+ and IgG1+ B cells with a memory phenotype outside of B cell follicles. After challenge, the IgG subset differentiated into plasmocytes, whereas the IgM subset reinitiated a germinal center reaction. This model, in which B cell memory appears in several layers with different functions, reconciles previous conflicting propositions.

Contribution of DNA polymerase η to immunoglobulin gene hypermutation in the mouse

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase η, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase ι gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of polη-deficient mice differs markedly. This suggests that, in the absence of polη, the MSH2–MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly polκ, to assume its function in hypermutation.

Mismatch Repair Deficiency Interferes with the Accumulation of Mutations in Chronically Stimulated B Cells and Not with the Hypermutation Process

Primary responses to the hapten phenyloxazolone and chronic responses to environmental antigens occurring in Peyers patches were analyzed in two different mismatch repair-deficient backgrounds. Paradoxically, whereas primary responses were found normal in MSH2- and only slightly diminished in PMS2-deficient mice, mutations in Peyers patch B cells from both k.o. animals were reduced three times, the subset of Peyers patch B cells with highly mutated sequences being specifically missing in the mismatch repair-deficient context. Strikingly, germinal center B cells from Peyers patches of k.o. animals showed microsatellite instability at an unprecedented level. We thus propose that the amount of DNA damages generated prevents these cells from recycling in germinal centers and that mismatch repair deficiency is only the indirect cause of the lower mutation incidence observed.

A Backup Role of DNA Polymerase κ in Ig Gene Hypermutation Only Takes Place in the Complete Absence of DNA Polymerase η

Patients with the variant form of xeroderma pigmentosum (XPV) syndrome have a genetic deficiency in DNA polymerase (Pol) η, and display accordingly an increased skin sensitivity to UV light, as well as an altered mutation pattern of their Ig V genes in memory B cells, alteration that consists in a reduced mutagenesis at A/T bases. We previously suggested that another polymerase with a different mutation signature, Pol κ, is used as backup for Ig gene hypermutation in both humans and mice in cases of complete Pol η deficiency, a proposition supported in this study by the analysis of Pol η × Pol κ double-deficient mice. We also describe a new XPV case, in which a splice site mutation of the first noncoding exon results in a decreased mRNA expression, a mRNA that otherwise encodes a normal Pol η protein. Whereas the Pol η mRNA level observed in patient’s fibroblasts is one-twentieth the value of healthy controls, it is only reduced to one-fourth of the normal level in activated B cells. Memory B cells from this patient showed a 50% reduction in A/T mutations, with a spectrum that still displays a strict Pol η signature. Pol η thus appears as a dominant enzyme in hypermutation, its presence precluding the use of a substitute enzyme even in conditions of reduced availability. Such a dominant behavior may explain the lack of Pol κ signature in Ig gene mutations of some XPV patients previously described, for whom residual Pol η activity might exist.

Competitive repair pathways in immunoglobulin gene hypermutation

This review focuses on the contribution of translesion DNA polymerases to immunoglobulin gene hypermutation, in particular on the roles of DNA polymerase eta (Polη) in the generation of mutations at A/T bases from the initial cytosine-targeted activation-induced cytidine deaminase (AID)-mediated deamination event, and of Polκ, an enzyme of the same polymerase family, used as a substitute when Polη is absent. The proposition that the UNG uracil glycosylase and the MSH2–MSH6 mismatch recognition complex are two competitive rather than alternative pathways in the processing of uracils generated by AID is further discussed.

Normal Immune System Development in Mice Lacking the Deltex-1 RING Finger Domain

ABSTRACT The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1Δ/Δ mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.

Ig gene hypermutation: A mechanism is due

Publisher Summary This chapter presents a discussion on Ig gene hypermutation. The chapter reviews (1) the possible participation of some mismatch repair (MMR) components, the mammalian MutS-homologs, (2) the role of Ig gene transcription in the targeting of the process, (3) the occurrence of DNA breaks as specific priming events, (4) the involvement of an error-prone DNA polymerase and the emergence of several new candidate enzymes, and (5) a new partner, activation-induced cytidine deaminase whose molecular contribution is the subject of intense speculation. Several reports have shown that transcription and hypermutation are quantitatively correlated starting with the earlier observation that the hypermutation domain at the heavy chain locus extends over 1 kb downstream from the Ig promoter. From these observations, various models have been proposed in which the transcription complex—as it progresses through the V gene—induces an error-prone repair process. The field of hypermutation is crowded with experiments and models but a precise molecular description of the process is due. The first molecule that seems to be a major player in the process has been discovered after a cDNA subtraction designed to elucidate switch recombination and its putative properties generate many more questions than they bring straightforward explanations.

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.

DNA polymerase η is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse

Delbos et al. 2007. J. Exp. Med. doi:10.1084/jem.20062131 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20062131%26rft_id%253Dinfo%253Apmid%252F17190840%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%